This invention relates to nucleic acid amplification with subsequent hybridization on solid supports (gene chip on glass support) as its application. More specifically, it relates to novel nucleic acid probes for detecting human hepatitis B virus surface antigen mutant 145 (Glycine to Arginine) in serum samples.
Throughout this invention, various publications are referenced by Arabic numerals. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
The present invention concerns the specific detection from serum samples of human hepatitis B virus surface antigen mutant 145 (Glycine to Arginine), by polymerase chain reaction, using novel nucleic acid probes. The present invention could be applied to detection of such mutant by other means, in particular detection of differential fluorescent signals after hybridization of unknown human viral DNA samples with a specific nucleic acid probe that is immobilized on solid supports (i.e. glass).
Viral hepatitis is a systemic disease involving primarily the liver, with hepatitis B virus being mainly responsible for most cases of acute or chronic hepatitis.
Antigenic characterization of human hepatitis B virus derives from the complex protein found on the virus"" surface, namely hepatitis B virus surface antigen. The major antigenic epitope, designated as xe2x80x98axe2x80x99 and located from amino acid 124 to 147 of the hepatitis B virus surface antigen, is common to all hepatitis B virus. This xe2x80x98axe2x80x99 epitope is directly involved in inducing neutralizing antibodies against hepatitis B viral infection. Such induction can be achieved by immunizing individuals with commercial available vaccines, consisting of non-infectious subviral hepatitis B surface antigen particles. An acquired protection in humans against hepatitis B viral infection is generally indicated by the presence of an adequate amount of serum antibody to hepatitis B virus surface antigen (anti-HBs). There is also a concomitant decrease of the serum viral surface antigen. However, an increasing numbers of incidence of hepatitis B viral infection despite the serum anti-HBs have been reported. These are largely contributed by hepatitis B viral strains that carry mutations on the antigenic region of the viral surface antigen, and in particular on the xe2x80x98axe2x80x99 epitope. Such surface antigen mutants are of serious concern as they display reduced affinity for the neutralizing antibodies and able to replicate independently. The most common mutation among these vaccine-escape hepatitis B virus variants has been found at amino acid residue 145 (Glycine to Arginine) on the xe2x80x98axe2x80x99 epitope of the viral surface antigen. In immunized infants born to HBeAg positive mothers, for example, the mutation 145 (Glycine to Arginine) within the major hydrophilic region is the most common variant found in those who subsequently become infected despite adequate amount of protective anti-HBs antibodies. This particular mutant is also the most common variant found in orthotopic liver transplantation patients who succumbed to hepatitis B viral infection despite immunoprophylaxis using hepatitis B immunoglobulin. Significantly, this human hepatitis B virus surface antigen mutant 145 (Glycine to Arginine) is also a naturally occurring variant that has been detected worldwide. In Singapore, despite the fact that an active vaccination program has resulted in a significant decrease of acute hepatitis B infection and the incidence of primary hepatocellular carcinoma in the general population, cases of breakthrough viral infection have been detected. Many of them (twelve out of forty-one) carry the viral surface antigen mutation 145 (Glycine to Arginine).
The emergence of this replicative hepatitis B virus surface antigen mutant 145 (Glycine to Arginine) and its ability to escape detection using currently available reagents are of grave concern, because this mutant is infectious and has resulted in the development of acute hepatitis B in Europe as well as Singapore. Our latest data also point to an increasing incidence of quasispecies in Singapore population consisting of both wild type and surface antigen 145 (Glycine to Arginine) of hepatitis B virus. Although serum human hepatitis B viral DNA can be detected by standard liquid hybridization assay (Abbott Laboratories, U.S.A.), such commercial kits are not designed to distinguish wild type hepatitis B virus from variants carrying mutations on hepatitis B surface antigen. A rapid and simple detection method for this particular human hepatitis B virus surface antigen mutant 145 (Glycine to Arginine) would therefore be useful for its diagnosis, therapy and prevention.
One approach toward this goal would be to detect the specific nucleic acid sequence of the HBsAg mutant 145 (Glycine to Arginine) in serum samples by specific Polymerase Chain Reaction amplification. Specific oligonucleotides would need to be designed on the basis of various available HBsAg sequences. Current methods of nucleic acid and oligonucleotide identifications have problems of sensitivity and selectivity, and have disadvantages such as the tedious and cumbersome analysis of the amplification results requiring highly skill operators to carry out the analyses i.e. agarose gel, polyacrylamide gel and molecular cloning. Application by the sophisticated oligonucleotide-based chip (Gene Chip) technology can provide further improved accuracy and rapid diagnostic screening assay. Gene Chip technology is now making more efficient and easier to use tools possible for obtaining and evaluating genetic information. This technology can be used for a broad spectrum of applications and analysis, such as sequence analysis, genotyping and monitoring of gene expression.
First developed in the late 1980s as a concept to determining DNA sequence by hybridization, the Gene Chip technology has been used in various fields of medicine and pharmaceutical research. Usually immobilized on solid support such as glass, the probe sequences can be originated from different procedures. These include the photolithographic synthesis of 20-25-mer oligonucleotides onto silicon wafers (Affymetrix, Glaxo-Welcome), printing of 500-5000 nucleotide cDNAs onto glass chip or dotting of pre-synthesized specific oligonucleotides (via their chemically modified terminus) onto glass chip. For the purpose of detecting hepatitis B surface antigen mutants with high specificity, the limited number of possible mutation sites (amino acid 100-160) would favor the dotting of pre-made oligonucleotides onto glass support in the application of Gene Chip technology. A specific detection system for hepatitis B surface antigen Glycine-145-Arginine mutant has been developed and described in this disclosure. It is based on novel nucleic acid probes which constitute an important innovative step towards the goals mentioned. Their identification would contribute to the effective prevention and control of hepatitis B viral infection arising from these viral surface antigen mutants, through rapid screening in blood banks, commercial and research diagnostic laboratories. This know-how information can also be used to detect other hepatitis B mutants.
The specific detection system described in the present invention that is based on novel nucleic acid probes constitutes an important step towards these goals and should contribute to the effective control of hepatitis B viral infection arising from these viral surface antigen mutants.
This invention describes novel nucleic acid probes that can be used in polymerase chain reaction to amplify specifically human hepatitis B surface antigen mutant 145 (Glycine to Arginine) from serum samples. In contrast to commercial liquid hybridization assays, the detection system in the present invention is able to detect the said human hepatitis B viral mutant from serum samples with specificity. The specificity of the present invention in turn allows detection of such mutant using gene chip technology, wherein the specific fluorescent labeled nucleic acid probe is immobilized on solid glass supports prior to hybridization reaction with target viral DNA fragment labeled by different fluorescent dye, and amplified from serum samples. The specific detection of this infectious human vaccine-escape hepatitis B surface antigen mutant 145 (Glycine to Arginine), from quasispecies serum samples, provides useful information for future monitoring of such mutants using specific therapeutic vaccines and effective antiviral agents.